Induction of intrinsic and extrinsic apoptosis through oxidative stress in drug-resistant cancer by a newly synthesized Schiff base copper chelate

Multidrug resistance (MDR) in cancer represents a variety of strategies employed by tumor cells to evade the beneficial cytotoxic effects of structurally different anticancer drugs and thus confers impediments to the successful treatment of cancers. Efflux of drugs by MDR protein-1, functional P-glycoprotein and elevated level of reduced glutathione confer resistance to cell death or apoptosis and thus provide a possible therapeutic target for overcoming MDR in cancer. Previously, we reported that a Schiff base ligand, potassium-N-(2-hydroxy 3-methoxy-benzaldehyde)-alaninate (PHMBA) overcomes MDR in both in vivo and in vitro by targeting intrinsic apoptotic/necrotic pathway through induction of reactive oxygen species (ROS). The present study describes the synthesis and spectroscopic characterization of a copper chelate of Schiff base, viz., copper (II)-N-(2-hydroxy-3-methoxy-benzaldehyde)-alaninate (CuPHMBA) and the underlying mechanism of cell death induced by CuPHMBA in vitro. CuPHMBA kills both the drug-resistant and sensitive cell types irrespective of their drug resistance phenotype. The cell death induced by CuPHMBA follows apoptotic pathway and moreover, the cell death is associated with intrinsic mitochondrial and extrinsic receptor-mediated pathways. Oxidative stress plays a pivotal role in the process as proved by the fact that antioxidant enzyme; polyethylene glycol conjugated-catalase completely blocked CuPHMBA-induced ROS generation and abrogated cell death. To summarize, the present work provides a compelling rationale for the future clinical use of CuPHMBA, a redox active copper chelate in the treatment of cancer patients, irrespective of their drug-resistance status.     

Silencing PLOD2 attenuates cancer stem cell-like characteristics and cisplatin-resistant through Integrin β1 in laryngeal cancer

Laryngeal cancer (LC) is an aggressive malignancy resistant to drug treatments. It has been postulated that cancer stem cells (CSCs) persist in a unique population of cancer cells involved in tumor progression and drug-resistance. In the present study, the effects of PLOD2 expression on ordinary and Cisplatin (DDP)-resistance (R) cells were investigated in TU686 and TU138 cells and Xenograft model. Cell viability, invasion and cell apoptosis, CD44 and CD133 expressions, MRP1 and P-gp expressions were measured by CCK-8 assay, Transwell, flow cytometry, immunofluorescence and Western blotting respectively. The results of our study demonstrated that suppressing the expression of PLOD2 could meditate LC stem cell-like features by decrease cell viability and invasion, increase apoptotic rate, decrease CD44 and CD133 expressions via Integrin β1. Meanwhile, the inhibition of PLOD2 expression could decrease P-gp and MRP1expression thus markedly regulate DDP-R LC cells stemness and drug-resistance via Integrin β1. Our findings provided a new rationale for subsequent academic and clinical research on LC drug-resistance.     

长春新碱诱导建立人结肠癌多药耐受性小鼠模型及MDR1和MRP1基因表达

目的建立人结肠癌多药耐受性动物模型并初步探索其耐药机制。方法结合体内外诱导方法建立人结肠癌多药耐受性动物模型,利用VCR和CTX的肿瘤抑制实验评价其MDR特性;利用real-time PCR和West-ern blotting等方法分析其P-gp/MDR1和MRP1基因和蛋白的表达。结果肿瘤抑制实验结果显示,MDR和敏感型结肠癌模型的肿瘤生长速度差异不显著,MDR结肠癌动物模型对于VCR和CTX的耐药性均有较大程度的提高;表达分析结果显示,人结肠癌MDR动物模型的P-gp/MDR1表达水平有较大提高,而MRP1表达没有显著变化。结论人结肠癌多药耐受性动物模型具有较好的多药耐受性,其多药耐受性表型主要是由于P-gp/MDR1过量表达所导致。     

Infection of H69AR cells with retroviral particles harboring interfering RNAi significantly reduced the multidrug resistance of these small cell lung cancer cells

Incubation of the drug-sensitive H69, a small cell lung cancer cell line, with increased concentrations of adriamycin yielded multidrug resistant (MDR) H69AR cells that over-express multidrug resistance-associated protein (MRP1). MRP1 co-transports its substrate with glutathione (GSH), leading to lower intracellular GSH. In this report we tested whether depleting intracellular GSH in MRP1-expressing cells could hyper-sensitize them to anticancer drugs or not. We have found that the GSH contents in MRP1-expressing cells are significantly lower than their corresponding control cells. The treatment with MRP1 substrate verapamil or the GSH synthetase inhibitor buthionine sulfoxi-mine significantly reduced the intracellular GSH contents in MRP1-expressing cells. Interestingly, depleting intracellular GSH contents can hyper-sensitize the MRP1-cDNA transfected BHK cells to daunomycin, but not the adriamycin-selected H69AR cells. Further analyses indicated that anti-apoptotic factor Bcl2 might be a factor responsible for the fact that depleting intracellular GSH could not hyper-sensitize H69AR cells to daunomycin. We hypothesized that knocking down the expression of Bcl2 could hyper-sensitize H69AR cells to daunomycin. Interestingly, infection of H69AR cells with retroviral particles harboring Bcl2 interfering RNAi not only reduced the expression of Bcl2, but also many factors that contribute to MDR, such as Bcl-xl, MRP1 and ABCC3, etc., leading to the MDR H69AR cells more sensitive to daunomycin than the parental H69 cell. Thus, although the mechanisms of the down-regulation of the genes contributing to MDR remain to be elucidated, retroviral particles harboring Bcl2 interfering RNAi could be used as an alternative way to sensitize the MDR cancer cells to anticancer drugs.     

Increased sensitivity to cis-diamminedichloroplatinum induced apoptosis with mitochondrial DNA depletion

Malignant cells harbor mechanisms which allow escape from drug-induced apoptosis, and the drug-resistance phenotype can be significantly associated with resistance to programmed cell death. There is accumulating evidence that mitochondria play a role in the tumorigenic phenotype, including the relative resistance to apoptosis. Whether changes at the mitochondrial level per se, would impact on the relative sensitivity of malignant cells to undergo drug-induced apoptosis, is not know. Accordingly, we determined if depleting mitochondrial DNA (mtDNA) would change the susceptibility of U937 cells to undergo apoptosis. With depletion, increases in sensitivity to cis-diamminedichoroplatinum (cisplatin)-induced apoptosis was observed. This sensitivity could be reverted to the parental phenotype by transforming the depleted cells with normal platelet mitochondria. mRNA expression of BAX, BCL2, MDR1, MRP, ERCC1 and ERCC2, putatively associated with cisplatin resistance to apoptotic death was unchanged. Inhibition of mitochondrial ATP production by oligomycin did not result in a change in ATP levels, indicating energetics were not playing a role in the observed phenotype changes. All U937 cells (with/without mtDNA) continued to respond to cisplatin by an apoptotic death. MtDNA-encoded molecules may be playing a role in the relative sensitivity of cells to undergo a cisplatin-induced apoptotic death, but may not be required for cells to undergo apoptosis per se.     

Targeting Mitochondrial Cell Death Pathway to Overcome Drug Resistance with a Newly Developed Iron Chelate

Background

Multi drug resistance (MDR) or cross-resistance to multiple classes of chemotherapeutic agents is a major obstacle to successful application of chemotherapy and a basic problem in cancer biology. The multidrug resistance gene, MDR1, and its gene product P-glycoprotein (P-gp) are an important determinant of MDR. Therefore, there is an urgent need for development of novel compounds that are not substrates of P-glycoprotein and are effective against drug-resistant cancer.

Methodology/Principal Findings

In this present study, we have synthesized a novel, redox active Fe (II) complex (chelate), iron N- (2-hydroxy acetophenone) glycinate (FeNG). The structure of the complex has been determined by spectroscopic means. To evaluate the cytotoxic effect of FeNG we used doxorubicin resistant and/or sensitive T lymphoblastic leukemia cells and show that FeNG kills both the cell types irrespective of their MDR phenotype. Moreover, FeNG induces apoptosis in doxorubicin resistance T lymphoblastic leukemia cell through mitochondrial pathway via generation reactive oxygen species (ROS). This is substantiated by the fact that the antioxidant N-acetyle-cysteine (NAC) could completely block ROS generation and, subsequently, abrogated FeNG induced apoptosis. Therefore, FeNG induces the doxorubicin resistant T lymphoblastic leukemia cells to undergo apoptosis and thus overcome MDR.

Conclusion/Significance

Our study provides evidence that FeNG, a redox active metal chelate may be a promising new therapeutic agent against drug resistance cancers.     

Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release

The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.     

Breast Cancer-Derived Microparticles Display Tissue Selectivity in the Transfer of Resistance Proteins to Cells

Microparticles (MPs) play a vital role in cell communication by facilitating the horizontal transfer of cargo between cells. Recently, we described a novel “non-genetic” mechanism for the acquisition of multidrug resistance (MDR) in cancer cells by intercellular transfer of functional P-gp, via MPs. MDR is caused by the overexpression of the efflux transporters P-glycoprotein (P-gp) and Multidrug Resistance-Associated Protein 1 (MRP1). These transporters efflux anticancer drugs from resistant cancer cells and maintain sublethal intracellular drug concentrations. By conducting MP transfer experiments, we show that MPs derived from DX breast cancer cells selectively transfer P-gp to malignant MCF-7 breast cells only, in contrast to VLB₁₀₀ leukaemic cell-derived MPs that transfer P-gp and MRP1 to both malignant and non-malignant cells. The observed transfer selectivity is not the result of membrane restrictions for intercellular exchange, limitations in MP binding to recipient cells or the differential expression of the cytoskeletal protein, Ezrin. CD44 (isoform 10) was found to be selectively present on the breast cancer-derived MPs and not on leukaemic MPs and may contribute to the observed selective transfer of P-gp to malignant breast cells observed. Using the MCF-7 murine tumour xenograft model we demonstrated the stable transfer of P-gp by MPs in vivo, which was found to localize to the tumour core as early as 24 hours post MP exposure and to remain stable for at least 2 weeks. These findings demonstrate a remarkable capacity by MPs to disseminate a stable resistant trait in the absence of any selective pressure.     

Redox active copper chelate overcomes multidrug resistance in T-lymphoblastic leukemia cell by triggering apoptosis

Multidrug resistance (MDR) mediated by the over expression of drug efflux protein P-glycoprotein (P-gp) is one of the major impediments to successful treatment of cancer. P-gp acts as an energy-dependent drug efflux pump and reduces the intracellular concentration of structurally unrelated drugs inside the cells. Therefore, there is an urgent need for development of new molecules that are less toxic to normal cell and preferentially effective against drug resistant malignant cells. In this preclinical study we report the apoptotic potential of copper N-(2-hydroxyacetophenone) glycinate (CuNG) on doxorubicin resistant T lymphoblastic leukaemia cells (CEM/ADR5000). To evaluate the cytotoxic effect of CuNG, we used different normal cell lines (NIH 3T3, Chang liver and human PBMC) and cancerous cell lines (CEM/ADR5000, parental sensitive CCRF-CEM, SiHa and 3LL) and conclude that CuNG preferentially kills cancerous cells, especially both leukemic cell types irrespective of their MDR status, while leaving normal cell totally unaffected. Moreover, CuNG involves reactive oxygen species (ROS) for induction of apoptosis in CEM/ADR5000 cells through the intrinsic apoptotic pathway. This is substantiated by our observation that antioxidant N-acetyle-cysteine (NAC) and PEG catalase could completely block ROS generation and, subsequently, abrogates CuNG induced apoptosis. On the other hand, uncomplexed ligand N-(2-hydroxyacetophenone) glycinate (NG) fails to generate a significant amount of ROS and concomitant induction of apoptosis in CEM/ADR5000 cells. Therefore, CuNG induces drug resistant leukemia cells to undergo apoptosis and proves to be a molecule having therapeutic potential to overcome MDR in cancer.     

Glutathione export during apoptosis requires functional multidrug resistance-associated proteins

GSH is released in cells undergoing apoptosis, and the present study indicates that the multidrug resistance-associated proteins (MRPs/ABCC) are responsible for this GSH release. Jurkat cells released approximately 75-80% of their total intracellular GSH during both Fas antibody- and staurosporine-induced apoptosis. In contrast, Raji cells, a lymphocyte cell line that is deficient in phosphatidylserine externalization, did not release GSH during apoptosis, and other apoptotic features appeared more slowly in these cells. Jurkat and Raji cell lines expressed comparable MRP and OATP/SLCO (organic anion-transporting polypeptide) mRNA levels, and MRP1 protein levels; however, differences existed in MRP1 localization and function. In Jurkat cells, MRP1 was largely localized to the plasma membrane, and these cells exported the MRP substrate calcein. Calcein release was enhanced during apoptosis. In contrast, Raji cells had little MRP1 at the plasma membrane and did not export calcein under basal or apoptotic conditions, indicating that these cells lack functional MRPs at the plasma membrane. GSH release in Jurkat cells undergoing apoptosis was inhibited by the organic anion transport inhibitors MK571, sulfinpyrazone, and probenecid, supporting a role for the MRP transporters in this process. Furthermore, when MRP1 expression was decreased with RNA interference, GSH release was lower under both basal and apoptotic conditions, providing direct evidence that MRP1 is involved in GSH export.     

Formononetin potentiates epirubicin-induced apoptosis via ROS production in HeLa cells in vitro

The frequent development of multidrug resistance (MDR) hampers the efficacy of available anticancer drugs in treating cervical cancer. In this study, we aimed to use formononetin (7-hydroxy-4′-methoxyisoflavone), a potential herbal isoflavone, to intensify the chemosensitivity of human cervical cancer HeLa cells to epirubicin, an anticancer drug. The reactive oxygen species (ROS) levels were correlated with MDR modulation mechanisms, including the transporter inhibition and apoptosis induction. Our results revealed that formononetin significantly enhanced the cytotoxicity of epirubicin. Co-incubation of epirubicin with formononetin increased the ROS levels, including hydrogen peroxide and superoxide free radicals. Epirubicin alone markedly increased the mRNA expression of MDR1, MDR-associated protein (MRP) 1, and MRP2. In contrast, formononetin alone or combined treatment decreased the mRNA expression of MRP1 and MRP2. This result indicates that efflux transporter-mediated epirubicin resistance is inhibited at different degrees by the addition of formononetin. This isoflavone significantly intensified epirubicin uptake into HeLa cells. Apoptosis was induced by formononetin and/or epirubicin, as signified by nuclear DNA fragmentation, chromatin condensation, increased sub-G1 and G2/M phases. The cotreatment triggered the mitochondrial apoptotic pathway indicated by increased Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, and significant activation of caspase-9 and -3. In addition, extrinsic/caspases-8 apoptotic pathway was also induced by the cotreatment. N-acetyl cysteine abrogated these events induced by formononetin, supporting the involvement of ROS in the MDR reversal mechanism. This study pioneered in demonstrating that formononetin may potentiate the cytotoxicity of epirubicin in HeLa cells through the ROS-mediated MRP inhibition and concurrent activation of the mitochondrial and death receptor pathways of apoptosis. Hence, the circumvention of pump and non-pump resistance using formononetin and epirubicin may pave the way for a powerful chemotherapeutic regimen for treating human cervical cancer.     

Regulatory mechanism of ZNF139 in multi-drug resistance of gastric cancer cells

Our previous study found increased zinc finger protein 139 (ZNF139) expression in gastric cancer (GC) cells. Purpose of the study is to further clarify the role and mechanism of ZNF139 in multi-drug resistance (MDR) of GC cells. MTT assay, RT-PCR, Western blotting were employed to detect susceptibility of GC cells to chemotherapeutic agents (5-FU, L-OHP) in vitro, and expressions of ZNF139 and MDR associated genes MDR1/P-gp, MRP1, Bcl-2, Bax were also detected. siRNA specific to ZNF139 was transfected into MKN28 cells, then chemosensitivity of GC cells as well as changes of ZNF139 and MDR associated genes were detected. It’s found the inhibition rate of 5-FU, L-OHP to well-differentiated GC tissues and cell line was lower than that in the poorly differentiated tissues and cell line; expressions of ZNF139 and MDR1/P-gp, MRP1 and Bcl-2 in well-differentiated GC tissues and cell line MKN28 were higher, while Bax expression was lower. After ZNF139-siRNA was transfected into MKN28, ZNF139 expression in GC cells was inhibited by 90 %; inhibition rate of 5-FU, L-OHP to tumor cells increased, and expressions of MDR1/P-gp, MRP1 and Bcl-2 were down-regulated, while Bax was up-regulated. ZNF139 was involved in GC MDR by promoting expressions of MDR1/P-gp, MRP1 and Bcl-2 and inhibiting Bax simultaneously.     

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.     

The role of a novel copper complex in overcoming doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

One of the important pathways of resistance to anthracyclines is governed by elevated levels of glutathione (GSH) in cancer cells. Resistant cells having elevated levels of GSH show higher expression of multidrug-resistant protein (MRP); the activity of glutathione S-transferases (GSTs) group of enzymes have also been found to be higher in some drug-resistant cells. The general mechanism in this type of resistance seems to be the formation of conjugates enzymatically by GSTs, and subsequent efflux by active transport through MRP (MRP1-MRP9). MRPs act as drug efflux pump and can also co-transport drugs like doxorubicin (Dox) with GSH. Depletion of GSH in resistant neoplastic cells may possibly sensitize such cells, and thus overcome multidrug resistance (MDR). A number of resistance modifying agents (RMA) like DL-buthionine (S, R) sulfoxamine (BSO) and ethacrynic acid (EA) moderately modulate resistance by acting as a GSH-depleting agent. As most of the GSH-depleting agents have dose-related toxicity, development of non-toxic GSH-depleting agent has immense importance in overcoming MDR. The present study describes the resistance reversal potentiality of novel copper complex, viz., copper N-(2-hydroxy acetophenone) glycinate (CuNG) developed by us in Dox-resistant Ehrlich ascites carcinoma (EAC/Dox) cells. CuNG depletes GSH in resistant (EAC/Dox) cells possibly by forming conjugate with it. Depletion of GSH results in higher Dox accumulation that may lead to enhanced rate of apoptosis in EAC/Dox cells. In vivo studies with male Swiss albino mice bearing ascitic growth of EAC/Dox showed tremendous increase in life span (treated/control, T/C = 453%) for the treated group with apparent regression of tumor. Resistance to Dox in EAC/Dox cells is associated with over expression of GST-P1, GST-M1 (enzymes involved in phase II detoxification) and MRP1 (a transmembrane ATPase efflux pump for monoglutathionyl conjugates of xenobiotics). CuNG causes down regulation of all these three proteins in EAC/Dox cells. The effect of CuNG as RMA is better than BSO in many aspects.     

Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

Multidrug resistance-associated protein 1 (MRP1) reduces intracellular anticancer drug accumulation either by co transporting them with glutathione (GSH) or extruding drug-GSH conjugates outside of the cell. Thus, MRP1 confers multidrug resistance (MDR) and worsen successful chemotherapeutic treatment against cancer. Although the exact mechanism of MRP1 involved in MDR remains unknown, the elevated level of intracellular GSH is considered as a key factor responsible for MDR in cancer. Hence the quest for non-toxic molecules that are able to deplete intracellular GSH has profound importance to subdue MDR. The present preclinical study depicts the resistance reversal potentiality of an iron complex; viz. Ferrous N-(2-hydroxy acetophenone) glycinate (FeNG) developed by us in doxorubicin resistant Ehrlich ascites carcinoma (EAC/Dox) cells. FeNG potentiate cytotoxic effect of doxorubicin on EAC/Dox cells ex vivo and also increases the survivability EAC/Dox bearing Swiss albino mice in vivo as well. Moreover, in vivo administration of FeNG significantly depletes intracellular GSH with ensuant increase in doxorubicin concentration in EAC/Dox cells without alternation of MRP1 expression. In addition, intra-peritoneal (i.p.) application of FeNG in normal or EAC/Dox bearing mice does not cause any systemic toxicity in preliminary trials in mouse Ehrlich ascites carcinoma model. Therefore, the present report provides evidence that FeNG may be a promising new resistance modifying agent against drug resistant cancers.     

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [³H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [³H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [¹²⁵I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.     

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc

In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.